Great news! No amplification in the 08:4-1 (positive for WS, but naive for phage)!! These results strongly suggest that these primers are specific for the WS bacteriophage! This is really cool and exciting. Next steps will be to confirm via in-situ hybridization (ISH).

Graphs were generated using the BioRad CFX Manager v2.0 software. Expression was normalized to EF1. Also to note, gene efficiency was assumed as 100% by the software since no standard curve was run on the plate. As such, analysis of this data may not be exact.

It’s clear by examining the graphs that the primers being used to differentiate COX1 and COX2 (since they share a common primer: SRID 1192) are differentially expressed. This indicates that the primer sets are indeed amplifying different targets as hoped. This was the primary intention of this qPCR. However, we also now have an idea of tissue distribution of the two genes, as well as their response to V. vulnificus exposre after 1hr. Next step is to perform this qPCR on all the individuals from this experiment as well as the different tissues.

mRNA was isolated for SOLiD sequencing by HTGU. Made two pools of San Nick RNA (Control and Exposed) using equal amounts (5ug) of each individual sample. Individual samples used can be found here. mRNA was isolated using Ambion’s Micro PolyAPurist Kit according to protocol. Procedure was performed two times on each pool and then EtOH precipitated. Samples were resuspended in 10uL of The RNA Storage Solution provided in the kit and spec’d on the Roberts Lab ND1000. Samples were stored @ -80C in the “Next Gen Sequencing Libraries” box.

Results:

Yields are pretty good from both samples (~500ng). However, the OD260/280 values are rather poor.

5ug of RNA was DNased using Ambion’s Turbo DNA-free kit, following the rigorous protocol (0.5uL of DNase for 30 mins then additional 0.5uL of DNase for 30mins). Calcs for DNase reactions are here. RNA was stored @ -80C in Shellfish RNA Boxes 4 and 5. Samples will be spec’d on Monday.

Results:

Overall, the RNA looks really good (based on OD 260/280 numbers). Not surprisingly, the OD 260/230 values for all samples dropped, likely due to the addition of the buffer (salts) used in the DNase reaction. Emma says she will check these samples for residual DNA.

–UPDATE (20110131)– Emma checked all DNased RNA samples on 20110131 using C.gigas 18s primers (SR ID: ?). She has not listed the results of the whether or not all samples are clean or if some still have residual gDNA carryover.

Used new cyclooxygenase primers (SR IDs 1060, 1061) to see how they performed and to evaluate tissue distribution. Tissue distribution was evaluated using the following cDNAs made on 10/27/10 from Emma:

Amplification is present in all four tissue types and the melting curve looks good. So, these primers are good to go. Steven suggests checking to see if we see a change in gene expression from an old experiment of Gigas exposed to high levels of Vibrio tubiashii. Will round up some old cDNA for this.

Ran qPCR on gDNA (06:50-10) to test new primers (H.iris_actin_intron_Fw/Rv) designed to bind only to a region in an intron of the H.iris actin gene. Hopefully there’s enough homology between H.iris (primer source) and H.cracherodii (template source) for this to work. PCR setup/plate layout is here. Anneal temp 50C.